Development of a TaqMan minor groove binding protein assay for the detection and quantification of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus and the family Bunyaviridae. It is a negative-strand RNA virus comprised of small (S), medium (M), and large (L) genome segments. The S segment encodes for nucleocapsid protein, the M segment codes for envelope glycoproteins (Gn and Gc), and the L segment codes for the RNA-dependent RNA polymerase. Currently, there are a limited number of methods for rapidly diagnosing CCHFV infections. We developed a real-time, reverse transcription-polymerase chain reaction assay for the rapid detection of CCHFV by using the TaqMan((R))-minor groove binding protein probe technology. The primers and probes were designed to amplify and detect a region in the S segment of CCHFV that is conserved across multiple strains. The limit of detection of the assay was 10 genome copies of RNA. This primer and probe set was specific to 18 strains of CCHFV tested and did not cross-react with either a DNA panel of 78 organisms or a panel of 28 diverse RNA viruses. This will rapidly and specifically detect CCHFV, and it has been used to detect CCHFV infection in samples from humans, animals, and ticks.

Laboratory transmission of Japanese encephalitis and West Nile viruses by molestus form of Culex pipiens (Diptera: Culicidae) collected in Uzbekistan in 2004.

We evaluated the molestus form of Culex pipiens pipiens (L.) (hereafter referred to as "molestus") captured near Tashkent, Uzbekistan, for their ability to transmit Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV) and West Nile (family Flaviviridae, genus Flavivirus, WNV) viruses under laboratory conditions. These molestus were highly competent laboratory vectors of WNV, with infection and dissemination rates of 96 and 81%, respectively. Approximately 75% of female molestus that fed after development of a disseminated infection transmitted virus by bite. Therefore, approximately 60% of those molestus taking a second bloodmeal between 16 and 25 d after an infectious bloodmeal would be expected to transmit WNV by bite. In contrast, these molestus were less efficient vectors of JEV, with infection and dissemination rates of 51 and 25%, respectively. In addition, only 33% of individuals with a disseminated infection transmitted JEV by bite, indicating a significant salivary gland barrier. Therefore, only approximately 8% of orally exposed individuals would be expected to transmit JEV by bite if they took a second bloodmeal 16-25 d later. These data indicate that the molestus form of Cx. p. pipiens should be considered a potentially important vector of WNV in Uzbekistan and may become involved in the transmission of JEV, should this virus be introduced into Uzbekistan.

Randomized, placebo-controlled trial of nonpegylated and pegylated forms of recombinant human alpha interferon 2a for suppression of dengue virus viremia in rhesus monkeys.

Dengue fever and dengue hemorrhagic fever are caused by infection with any one of the four dengue viruses (DVs) and are significant public health burdens throughout the tropics. Higher viremia levels are associated with greater dengue disease severity. A therapeutic intervention to suppress viremia early in DV infection could potentially ameliorate severe disease. Recombinant alpha interferon 2a (rIFN-alpha-2a, Roferon-A) suppressed DV replication in human peripheral blood mononuclear cells in vitro. We therefore examined the effects of rIFN-alpha-2a and pegylated recombinant IFN-alpha-2a (PEG-rIFN-alpha-2a, PEGASYS) on DV serotype 2 (DV-2) viremia in rhesus monkeys. Flavivirus-naïve monkeys were inoculated with DV-2 and randomized to receive a single dose of rIFN-alpha-2a (10 million international units/m2) versus placebo or PEG-rIFN-alpha-2a (6 microg/kg) versus placebo 1 day after the onset of viremia. Serial daily viremia levels were measured, and convalescent-phase DV-2 neutralizing antibody titers were determined. Compared to placebo, a single injection of rIFN-alpha-2a temporarily suppressed DV-2 replication and delayed the time to peak viremia by a median of 3 days. However, measures of total viral burden were not different between the two groups. A single injection of PEG-rIFN-alpha-2a significantly lowered daily viremia levels and improved virus clearance, starting 48 h after administration. There were no significant differences in DV-2 neutralizing antibody titers between the treatment and placebo groups at 30 and 90 days postinfection. Based on their individual effects, future studies should investigate a combination of rIFN-alpha-2a and PEG-rIFN-alpha-2a for suppression of dengue virus viremia and as a potential therapeutic intervention.

Experimental transmission of Karshi and Langat (tick-borne encephalitis virus complex) viruses by Ornithodoros ticks (Acari: Argasidae).

Selected species of mosquitoes and Ornithodoros ticks were evaluated for their potential to transmit Karshi and Langat (tick-borne encephalitis virus complex) viruses in the laboratory. Although there was no evidence of replication of Karshi virus in either of the two mosquito species tested [Ochlerotatus taeniorhynchus (Wiedemann) or Culex pipiens (L.)], Karshi virus replicated in and was transmitted by all three species of Ornithodoros ticks tested (Ornithodoros parkeri Cooley, Ornithodoros sonrai Sautet & Witkowski, and Ornithodoros tartakovskyi Olenev). When inoculated with Karshi virus, 90% of Ornithodoros ticks (44/49) transmitted this virus by bite to suckling mice, and transmission continued to occur for at least 1 yr, the longest extrinsic incubation tested. After feeding on a suckling mouse with a viremia of approximately 10(5) suckling mouse subcutaneous lethal dose. units of Karshi virus per milliliter of blood, all three species of Ornithodoros tested became infected with and transmitted Karshi virus both trans-stadially and horizontally by bite to suckling mice. In addition, female O. tartakovskyi transmitted Karshi virus vertically to their progeny. In a continuation of a previous study, O. sonrai, orally exposed to Langat virus, were able to transmit this virus after >3 yr, the longest interval tested. Therefore, Ornithodoros spp. should be considered as potential vectors and as possible long-term maintenance hosts for Karshi virus and other members of the tick-borne encephalitis virus complex.

HLA-A and -B allele associations with secondary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais.

Little is known of the role of classical HLA-A and -B class I alleles in determining resistance, susceptibility, or the severity of acute viral infections. Appropriate paradigms for immunogenetic studies of acute viral infections are dengue fever (DF) and dengue hemorrhagic fever (DHF). Both primary and secondary infections with dengue virus (DEN) serotypes 1, 2, 3 or 4, can result in either clinically less severe DF or the more severe DHF. In secondary exposures, a memory response is induced in immunologically primed individuals, which can both clear the infecting dengue virus and contribute to its pathology. In a case-control study of 263 ethnic Thai patients infected with either DEN-1, -2, -3 or -4, we detected HLA class I associations with secondary infections, but not in immunologically naive patients with primary infections. HLA-A*0203 was associated with the less severe DF, regardless of the secondary infecting virus serotype. By contrast, HLA-A*0207 was associated with susceptibility to the more severe DHF in patients with secondary DEN-1 and DEN-2 infections only. Conversely, HLA-B*51 was associated with the development of DHF in patients with secondary infections, and HLA-B*52 was associated with DF in patients with secondary DEN-1 and DEN-2 infections. Moreover, HLA-B44, B62, B76 and B77 also appeared to be protective against developing clinical disease after secondary dengue virus infection. These results confirm that classical HLA class I alleles are associated with the clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals.

First documented outbreak of hepatitis E virus transmission in Java, Indonesia.

A suspected hepatitis outbreak occurred in Bondowoso District, East Java Province, Indonesia, in March-May 1998. An investigation was initiated in April 1998, involving a retrospective review of hospital records, a community-based cross-sectional study, and a health service-based case detection and household follow-up. Sera and epidemiological information were collected from 962 individuals: 235 from 3 outbreak-affected communities along the same rural stretch of river, 101 from community controls living distant from the river, 151 cases detected in health centres, 141 family members of the cases, and 334 subjects from neighbouring families. The prevalence of acute hepatitis E virus (HEV), based on anti-HEV IgM, total antibody (Ig) to HEV and polymerase chain reaction (PCR), was significantly (P < 0.00001) higher (52.4%) among the outbreak communities than among the community controls (3%). The background prevalence of HEV, based on anti-HEV IgG, was also significantly (P < 0.00001) higher (47%) among the outbreak communities than among the community controls (3%). None of the 476 sera screened for anti-HAV (hepatitis A virus) IgM was positive. These results indicate that HEV was the aetiological agent responsible for the outbreak. The overall attack rate (AR) for the 3 outbreak-affected communities surveyed was 19%, with AR determined on the basis of clinically recognized, acute jaundice illness. The usage of river water as primary source for bathing, human-waste disposal, and drinking purposes differed significantly (P < 0.00001) between the communities in outbreak areas and those in non-outbreak areas. There is no significant influence attributed to 'boiling water' on acute HEV. No climatic influences (flooding or drought) predisposed this instance of epidemic HEV transmission. This outbreak represents the first documented evidence of epidemic HEV transmission in Java, Indonesia.

Extinction and rapid emergence of strains of dengue 3 virus during an interepidemic period.

Strains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection.

Molecular serotyping of dengue viruses in field-caught Aedes mosquitos by in-house RNA extraction/RT-PCR reagent kits.

We developed in-house RNA extraction and RT-PCR reagent kits for the molecular serotyping of dengue viruses in field-caught Aedes mosquitos. Mosquitos that showed positive results by ELISA or IFA were selected for the identification of dengue viruses in order to predict the distribution of the four dengue serotypes. Total RNA was extracted from one whole mosquito as well as from one dissected mosquito by our in-house RNA extraction reagents using the modified method of guanidinium thiocyanate denaturation and isopropanol precipitation. The extracted RNA was amplified by our in-house RT-PCR reagents specific for each dengue serotype under optimized conditions. Dengue viral RNA extracted from a single mosquito as well as from the head and thorax of one dissected mosquito could be detected successfully; it could not be found in the abdomen, however. These results indicated that most of the dengue viruses were located in the head and thorax rather than in the abdomen. The results of dengue serotyping showed a pure specific PCR product for each dengue serotype at 490, 230, 320 and 398 bp for DEN-1, DEN-2, DEN-3, and DEN-4 respectively. In addition, the detection sensitivity was very high: an amount of RNA template equivalent to approximately 1/80 of a single mosquito could be detected by agarose gel electrophoresis and ethidium bromide staining. The coupling of RT-PCR-based surveillance of dengue viral infection with disease mapping data (Geograpical Information System, GIS) could serve as an alternative epidemiological means of providing an early warning of dengue fever/dengue hemorrhagic fever epidemics.

Use of recombinant envelope proteins for serological diagnosis of Dengue virus infection in an immunochromatographic assay.

An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay were 90 and 86%, respectively. The similar diagnostic performance of these antigens to that of enzyme-linked immunosorbent assays using whole dengue virus suggests that they mimic whole dengue viruses in primary structure and epitope conformation. These results suggest that recombinant proteins can be used in diagnostic assays for dengue to overcome safety issues associated with the use of whole virus.

Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis.

The ability of dendritic cells (DCs) to shape the adaptive immune response to viral infection is mediated largely by their maturation and activation state as determined by the surface expression of HLA molecules, costimulatory molecules, and cytokine production. Dengue is an emerging arboviral disease where the severity of illness is influenced by the adaptive immune response to the virus. In this report, we have demonstrated that dengue virus infects and replicates in immature human myeloid DCs. Exposure to live dengue virus led to maturation and activation of both the infected and surrounding, uninfected DCs and stimulated production of tumor necrosis factor alpha (TNF-alpha) and alpha interferon (IFN-alpha). Activation of the dengue virus-infected DCs was blunted compared to the surrounding, uninfected DCs, and dengue virus infection induced low-level release of interleukin-12 p70 (IL-12 p70), a key cytokine in the development of cell-mediated immunity (CMI). Upon the addition of IFN-gamma, there was enhanced activation of dengue virus-infected DCs and enhanced dengue virus-induced IL-12 p70 release. The data suggest a model whereby DCs are the early, primary target of dengue virus in natural infection and the vigor of CMI is modulated by the relative presence or absence of IFN-gamma in the microenvironment surrounding the virus-infected DCs. These findings are relevant to understanding the pathogenesis of dengue hemorrhagic fever and the design of new vaccination and therapeutic strategies.

TT virus infection in acute non-A to E hepatitis in northern Thailand.

TT virus is a novel DNA virus widely distributed in the general population. We examined the prevalence of TTV infection in a population with acute non-A to E hepatitis and in comparison groups located in Northern Thailand. The prevalence of TTV in subjects with non-A-E hepatitis was 19% (21/112), 6% (4/72) in healthy volunteers, 17% (12/72) in those with hepatitis A or B, and 17% (8/48) in hospitalized patients with non-hepatitis illnesses. A significant association with TTV infection and non-A-E hepatitis was seen in all groups (OR 3.9, p = 0.02) and in children (OR 25.8, p = 0.001). Among subjects with non-A-E hepatitis, TTV was associated with higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (significant for AST, p = 0.02). Our observations suggest that TTV in our study population may be associated with non-A-E hepatitis and that children in particular may be at risk of hepatocellular injury as a result of TTV infection.

Mechanisms of hemorrhage in dengue without circulatory collapse.

To characterize the molecular basis for the hemostatic defects of dengue infections, a study was conducted in Bangkok, Thailand. Febrile children (n = 68) hospitalized with suspected dengue were enrolled before their clinical syndromes were classified as either dengue fever (DF) or dengue hemorrhagic fever (DHF). Hospital course and outcome were recorded; blood was obtained during the febrile illness (S1), after defervescence (S2), and 1 month after onset of disease (S4). Patients were classified as DF (n = 21) and DHF grades 1, 2, and 3; (DHF1, n = 8; DHF2, n = 30; and DHF3, n = 9). All had marked thrombocytopenia. Bleeding scores were assigned on the basis of bleeding site. Although there was no correlation between bleeding scores and pleural effusion index (a measure of vascular leakage) or bleeding scores and platelet counts, there was a correlation between pleural effusion index and platelet counts. Bleeding scores did not correlate with hemostatic data. Activated partial thromboplastin time was prolonged, with trends toward decreased fibrinogen and increased levels of prothrombin fragment F1.2 in the acute-phase samples. However, no factor level was dramatically decreased. We conclude that most patients with DF or DHF, even without overt hemorrhage, have consumptive coagulopathy. Nevertheless, hemorrhage in dengue without circulatory collapse is most likely due to activation of platelets rather than coagulopathy, which is well compensated. Our data suggest that vascular alteration may be the principal factor involved in the association of thrombocytopenia and hemorrhage with disease severity.

Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity.

Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.

Evaluation of a new commercially available immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of Japanese encephalitis infections.

A new commercial enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Japanese encephalitis virus infections showed a sensitivity of 88% with sera and 81% with cerebrospinal fluid and a specificity of 97% with sera from patients with primary and secondary dengue virus infections. Specificity was 100% when samples from nonflavivirus infections were tested.

Safety and efficacy of intravenous sodium stibogluconate in the treatment of leishmaniasis: recent U.S. military experience.

The efficacy and toxicity of sodium stibogluconate (SSG) at a dosage of 20 mg/(kg.d) for either 20 days (for cutaneous disease) or 28 days (for visceral, mucosal, or viscerotropic disease) in the treatment of leishmaniasis is reported. Ninety-six U.S. Department of Defense health care beneficiaries with parasitologically confirmed leishmaniasis were prospectively followed for 1 year. One patient was infected with human immunodeficiency virus; otherwise, comorbidity was absent. Clinical cure occurred in 91% of 83 cases of cutaneous disease and 93% of 13 cases of visceral/viscerotropic disease. Adverse effects were common and necessitated interruption of treatment in 28% of cases, but they were generally reversible. These included arthralgias and myalgias (58%), pancreatitis (97%), transaminitis (67%), headache (22%), hematologic suppression (44%), and rash (9%). No subsequent mucosal leishmaniasis was identified, and there were no deaths attributable to SSG or leishmaniasis.

Acute jaundice in Vientiane, Lao People's Democratic Republic.

Analysis of serum samples from patients with acute jaundice by means of enzyme-linked immunosorbent assay and polymerase chain reaction testing provided the first profile of this condition in Vientiane, Lao PDR, in 1995 and 1996. In a case-control, hospital-based study, evidence of acute infections due to hepatitis A and B viruses was found in 14% and 10% of cases, respectively. Hepatitis E virus, however, did not appear to contribute to clinically recognized acute jaundice. Similarly, antibody to hepatitis C virus was recognized in almost equal proportions of cases (8%) and controls (6%), thus representing probable background infections. The detection of hepatitis G virus marks the first report of this virus in Lao PDR. The large proportion (21%) of new leptospiral infections in cases without acute hepatitis A or B was notable. This finding suggests significant regional underreporting of leptospirosis as a cause of acute jaundice. The limited laboratory diagnostic capabilities for confirming a differential diagnosis of leptospirosis contribute to the lack of attention paid to this important health problem.

Trimethoprim/sulfamethoxazole desensitization.

Although alternative therapy for PCP remains limited, the role of TMP/SMX desensitization becomes increasingly important in patients with AIDS. Various successful desensitization protocols have been described in this article. As there are no established guidelines, it appears that the desensitization procedure can occur in small successive doses given each day or one small dose given daily. An evaluation of the severity of allergic reaction can be used to determine the type of dosing regimen. We believe that protocols starting with low doses and slow titration to full-dose therapy, as used at our institution, should be efficacious. Monitoring of the patient after the desensitization procedure should continue, as sensitivity may reoccur. In addition, while the patient is undergoing desensitization, some investigators recommend that alternative therapy be continued until full-dose TMP/SMX therapy is achieved. Also, it is important to realize that once a patient is successfully desensitized, medication compliance must be maintained because, theoretically, reexposure to the drug after a lapse in therapy may result in hypersensitivity reactions. Therefore, this procedure and the possibility of serious adverse effects, such as Stevens-Johnson syndrome and anaphylaxis, should be evaluated carefully and discussed thoroughly with each patient prior to initiation of therapy. Finally, a study of sufficient size should be performed to evaluate the efficacy of desensitization regimens and establish specific dosing guidelines.